1. Field of the Invention
This invention relates generally to methods and devices for determining the percent inhibition of platelets, and more particularly to methods and devices for determining the percent inhibition of platelets when treated with drugs that suppress various activation pathways of platelets.
2. Description of the Related Art
The role of platelets in mammalian physiology is extraordinarily diverse, but their primary role is in promoting hemostasis. In many situations, an evaluation of the ability of blood to clot is desired, a parameter that is frequently controlled by the ability of platelets to adhere and/or aggregate. Of interest, therefore, is the assessment of the adhesive functions of platelets. For example, questions of interest include whether to administer drugs that will block, or promote, clot formation, or whether to detect deficiencies in platelet function prior to surgical procedures. Also of interest is evaluating the effectiveness of a platelet inhibitor that is being tested as a new drug or is being used as approved clinical treatment in a patient.
Platelets play a critical role in the maintenance of normal hemostasis. When exposed to a damaged blood vessel, platelets will adhere to exposed sub-endothelial matrix. Following the initial adhesion, various factors released or produced at the site of injury such as thrombin, ADP and collagen activate the platelets. Once platelets are activated, a conformational change occurs in the platelet glycoprotein GPIIb/IIIa receptor, allowing it to bind fibrinogen and/or von Willebrand factor. It is this binding of the multivalent fibrinogen and/or von Willebrand factor molecules by GPIIb/IIIa receptors on adjacent platelets that results in the recruitment of additional platelets to the site of injury and their aggregation to form a hemostatic plug or thrombus.
Platelet aggregation is a term used to describe the binding of platelets to one another. In vitro platelet aggregometry is the laboratory method used to assess the in vivo ability of platelets to form the aggregates leading to a primary hemostatic plug. In this technique an anti-coagulated whole blood sample is centrifuged under multiple conditions to create both a platelet-rich plasma (PRP) and platelet-poor plasma (PPP) sample. An aggregating agent such as ADP or collagen is then added to the PRP and aggregation of platelets monitored optically while in parallel with this, a separate optical measurement is made using the PPP sample. The percent aggregation is then determined by use of the PPP channel as the 100% aggregation reference level to compare with the PRP channel. Because the centrifugation of the blood can result in hemolysis of the red-blood cells, the change in optical density of the PPP due to preparation variability can result in errors in the optical reference level used to calculate the percent aggregation.
Another in vitro platelet aggregometry method used in the laboratory to assess in vivo platelet aggregation capability, uses diluted whole blood to measure the change in electrical impedance between two closely spaced precious metal electrodes as platelets adhere and aggregate after addition of an aggregating agent such as ADP, or collagen. With this method, there is no single assay means to determine the percent of platelet aggregation. Instead, two separate assays must be run and the ratio between the two measurements taken to determine the percent aggregation. For the case where the patient is already on a platelet inhibiting drug, there is no means of determining their percent of platelet aggregation. Current assays to measure platelet aggregation are expensive, time-consuming, cumbersome, and generally not suitable for a clinical environment.
A rapid platelet function assay has recently been developed and is described in U.S. Pat. No. 5,763,199. The assay determines glycoprotein (GP)IIb/IIIa receptor blockade in undiluted whole blood. Agglutination of small polymeric beads coated with a GPIIb/IIIa ligand such as fibrinogen results when the beads are contacted with whole blood containing platelets with activated GPIIb/IIIa receptors that are not blocked. Failure to agglutinate indicates either failure of the GPIIb/IIIa receptors to become activated and/or blockade of the GPIIb/IIIa receptors. In a preferred embodiment, the addition of a platelet activator like ADP or arachidonic acid, results in an assay that is rapid and convenient enough to be performed at the bedside and that results in agglutination of the small polymeric beads within a convenient, known period of time if the activation receptors are not blocked. The assay includes the ability to transfer blood to be tested from a collection container to an assay device without opening the collection container.
Platelet aggregation plays a key role in the pathogenesis of thrombosis and acute coronary artery disease. Evidence suggests that significant platelet function variability exists in the response to various antiplatelet agents. It has also been demonstrated that an inter-individual variability in platelet aggregation exists when P2Y12 antagonists such as clopidogrel are used for treatment of patients to achieve an anti-aggregation effect. The results of one study demonstrated that at least 10% of patients receiving the drug did not achieve the expected platelet aggregation inhibition (Muller I, Besta F, Schulz C, Massberg S, Schonig A, Gawaz M; Prevalence of clopidogrel non-responders among patients with stable angina pectoris scheduled for elective coronary stent placemen Thromb Haemost. 2003 May, 89(5):783-7).
Since many patients with cardiovascular disease are chronically taking one of the thienopyridine agents, a method for determining the level of platelet inhibition based on a single measurement of an inhibited sample would be beneficial. In addition, patients undergoing PTCA procedures are generally given a large bolus dose of a thienopyridine agent prior to the procedure. Some of these patients may then require emergent surgery and an assay that would provide information about their absolute level of platelet inhibition would be beneficial in assessing the bleeding management risks prior to surgery.
There is a need for an improved method for obtaining a percent aggregation or inhibition of platelets, due to anti-platelet drugs. There is a further need for a method for obtaining a percent aggregation or inhibition of platelets, due to anti-platelet drugs, using a single blood sample. There is yet a further need for a method for obtaining a percent aggregation or inhibition of platelets, due to anti-platelet drugs, in a single measurement.